RNA-seq of mouse Pancreatic Intraepithelial Neoplasias (PanINs) with NF-κB Essential Modulator (NEMO) knockout against NEMO wild-type
RNA-seq of mouse Pancreatic Intraepithelial Neoplasias (PanINs) with NF-κB Essential Modulator (NEMO) knockout against NEMO wild-type
Cryopreserved tissue was sectioned and placed in poly-L-lysine-coated membrane slides and air-dried for 5 minutes. Then, slides were incubated serially in 70% ethanol for 3 minutes, distilled water for 30 seconds, hemalum (Merck #109249, diluted 1:3 in distilled water) for one minute, distilled water for 30 seconds, 70% ethanol for 30 seconds, 90% ethanol for 30 seconds, 100% ethanol for 30 seconds and air-dried. Lesions were then excised and captured using the PALM Microbeam Rel. 4.9 with RoboMover microscope (Zeiss) and the PALMRobo v 4.9 software. Ethanol dilutions for the LCM preparation were stored at -20oC. RNAse-free water for the LCM preparation was stored at 4oC. RNA from isolated lesions was extracted with RNeasy Micro Kit (QIAGEN #74004) according to its standard protocol. RNAse-free water was used in all steps (including dilution of ethanol) to avoid RNA degradation. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligoattached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library.